Proposed research will critically test our new hypothesis that drug-protein adducts are causal factors in the pathogenesis of diclofenac-induced ulcers in the small intestine. This hypothesis stems from our novel observations that diclofenac forms adducts with enterocytes prior to ulceration and to a greater extent in the jejunal region most prone to ulceration. This classical NSAID is widely prescribed for arthritis, musculoskeletal injuries and pen-operative pain. Chronic use of classic NSAIDs, including diclofenac, is complicated by a >50 percent incidence of enteropathy (i.e., inflammation, lesions and blood loss) plus high risks of hospitalization and death secondary to intestinal perforation. Specific Aim I will characterize the hypothesized critical early sites of adduct formation utilizing immunohistochemistry, confocal microscopy, and 14C-diclofenac to localize the distribution and extent of adduction prior to structural alterations of enterocytes and villi in ulcer-prone regions of the intestine. A jejunum-to-jejunum anastamosis technique will assess alternative explanations for adduct accumulation at sites of ulcers. Specific Aim 2 will exploit regional difference in diclofenac-induced intestinal ulceration to define linkages between adduction and potentially relevant functional and chemical consequences. Experiments will determine if adduction of ulcer-prone regions is associated with impairments of vital functions or with evidence for oxidative stress. A temporal linkage between adduction and impaired capacity for repair by restitution will be specifically sought using a differentiated intestinal epithelial cell line provided by Dr Peter G Traber. A new collaboration with Dr Daniel Liebler will allow application of a novel proteomics approach to the identification of protein targets of diclofenac adduction. The sensitivity of the planned detection techniques, particularly the MS-MS fragmentation technique for adduct proteonomics, will enable analysis of tiny quantities of tissue obtained by microdissection or laser capture microdissection. Specific Aim 3 will critically test the purported role of oxidative stress by determining if the advancement to ulceration after adduct formation can be modified by intralumenal infusion of an antioxidant rescue or a lipid peroxide challenge. Current COX-inhibition based theories for NSAID-induced gastric ulcers do not account for the enteropathy problem. Therefore, information obtained from the proposed research about adducts as the initial molecular insult to enterocytes could provide a new mechanistic basis for development of intestine-safer NSAIDs or regimes of NSAID therapy.